Interactions of ANP and ANG II in tubular nephron acidification

Transcription

1 Brazilian Journal of Medical and Biological Research (1997) : and II in nephron acidification ISS X 471 Interactions of and II in tubular nephron acidification M. Mello-Aires, M.L.M. Barreto-haves, G. ascimento-gomes and M. Oliveira-Souza Departamento de Fisiologia e Biofísica, Instituto de iências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil orrespondence M. Mello-Aires Departamento de Fisiologia e Biofísica Instituto de iências Biomédicas Universidade de São Paulo São Paulo, SP Brasil Fax: 55 (011) Presented at the International Symposium euroendocrine ontrol of Body Fluid Homeostasis, Ribeirão Preto, SP, Brasil, August -20, Research supported by FAPESP, Pq and FIEP. Received ovember 29, 1996 Accepted January 6, 1997 Abstract In order to examine the effects and the interaction of angiotensin II ( II, 1 pm) and atrial natriuretic peptide (, 1 µm) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen ph by Sb microelectrodes. These studies confirmed that II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that alone does not affect this process, but impairs the stimulation caused by II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular ph was measured with a double-barreled microelectrode with H -sensitive resin. The results indicate that II acted by stimulating a /H exchange in ED (81%) and LD (54%) segments via activation of AT 1 receptors, as well as vacuolar H -ATPase in LD segments (33%). did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by II. To investigate the mechanism of action of these hormones in more detail, we studied cell ph dependence on II and in MDK cells using the fluorescent probe BEF. We showed that the velocity of cell ph recovery was almost abolished in the absence of a, indicating that it is dependent on a /H exchange. (1 µm) alone had no effect on this recovery but reversed both the acceleration of H extrusion at low II levels (1 pm and 1 nm), and inhibition of H extrusion at higher II levels (100 nm). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [a 2 ] i, monitored with the fluorescent probe Fura-2 in MDK cells in suspension. The data indicate that the addition of increasing concentrations of II (1 pm to 1 µm) to the cell suspension led to a progressive increase in [a 2 ] i to 2-3 times the basal level. In contrast, the addition of (1 µm) to the cell suspension led to a very rapid 60% decrease in [a 2 ] i and reduced the increase elicited by II, thus modulating the effect of II on [a 2 ] i. These results may indicate a role of [a 2 ] i in the regulation of the H extrusion process mediated by a /H exchange and stimulated/impaired by II. The data are compatible with stimulation of a /H exchange by increases of [a 2 ] i in the lower range, and inhibition at high [a 2 ] i levels. Key words Angiotensin II Bicarbonate reabsorption ell ph ell a 2

2 472 M. Mello-Aires et al. JHO 3 - (nmol cm -2 s -1 ) Luminal perfusion The atrial natriuretic peptides (s) are a family of peptides originating from mammalian heart atria which present strong natriuretic and diuretic properties. Several studies indicate that acts directly on the kidney by enhancing the glomerular filtration rate (GFR) (1-3). However, it remains unclear whether has a direct effect on the proximal tubule or whether the observed diuresis and natriuresis are the result of an increased fluid flow along this nephron segment caused by the increased GFR. Because several of the studies were performed in vivo and functional cholinergic receptors on the basolateral side of the proximal tubule can regulate bicarbonate reabsorption and fluid flux (4), systemic effects of and the influence of the renal nerves may mask a direct action of on the proximal nephron. Angiotensin II ( II), an octapeptide naturally found in blood, is a potent regulator of acidification in the rat early proximal tubule (5). This hormone stimulates both Figure 1 - Effect of atrial natriuretic peptide (, 1 µm) and/or angiotensin II (, 1 pm) on HO 3 - reabsorption in proximal tubule. The experiments were performed in tubules undergoing luminal (left) or peritubular capillary (right) perfusions with the hormones. Data are reported as means ± SEM., umber of microperfusions; J HO3 -, net HO 3 - reabsorption. P<0.05 compared to control (); P<0.05 compared to II (Bonferroni test). 44 Peritubular perfusion apical a /H exchange and basolateral a - HO 3 - cotransport in perfused S1 segments of proximal tubules isolated from superficial nephrons of the rabbit kidney (6). Electrophysiological studies of II regulation of a -HO 3 - cotransport in isolated perfused S2 segments of rabbit renal proximal tubules confirmed that picomolar concentrations of II stimulate (7), while micromolar concentrations inhibit (8) basolateral a -HO 3 - cotransport. An interaction between and II has been observed in a variety of tissues. inhibits the vasoconstrictor effect of II on blood vessels in vitro (9), as well as the systemic pressor action of II (10) and II-stimulated aldosterone synthesis (11). Using the split-droplet technique, Harris et al. (12) found that had an inhibitory effect on proximal fluid absorption stimulated by preliminary II perfusion. Similar results were obtained by Garvin (13) in isolated perfused proximal straight tubules. However, the same effect was not observed by Liu and ogan (14) during in vivo free-flow micropuncture experiments. In order to examine the effects and the interaction of II (1 pm) and (1 µm) on the kinetics of bicarbonate reabsorption in the middle proximal tubule of rats, we performed in vivo experiments using a stopped-flow microperfusion technique, which is not affected by GFR (15). In this preparation, the systemic effects of both hormones were eliminated during luminal or peritubular capillary perfusion, as confirmed by the absence of changes in urine flow and a excretion under these conditions. Bicarbonate reabsorption was measured by stationary microperfusion and lumen ph was determined with Sb microelectrodes. et bicarbonate reabsorption (J HO3 -) was obtained by the following relation: J HO3 - = k([ho 3 - ] i - [HO 3 - ] s ) r/2 where k is the rate constant of the reduction

3 and II in nephron acidification 473 of luminal bicarbonate [k = ln2/(t/2)], t/2 is the half-time of luminal bicarbonate disappearance, r is the tubule radius, and [HO 3 - ] i and [HO 3 - ] s are the concentrations of the injected HO 3 - and HO 3 - at the stationary level, respectively. Figure 1 shows some of the results obtained in this study. It was noted that perfused in the lumen did not change proximal bicarbonate reabsorption but inhibited the stimulation induced by luminal perfusion of II: J HO3 - was 2. ± 0.10 nmol cm -2 s -1 ( = 86) with luminal control perfusion, 2.18 ± 0.25 nmol cm -2 s -1 ( = 33) during luminal perfusion, increased to 3.94 ± 0.35 nmol cm -2 s -1 ( = 35) with II luminal perfusion and decreased to 2.21 ± 0.15 nmol cm -2 s -1 ( = 46) during II plus luminal perfusion. The perfusion of in the peritubular capillary also did not affect proximal bicarbonate reabsorption but caused an inhibition of the stimulatory effect of II in peritubular capillary perfusion: J HO3 - was 1.83 ± 0.14 nmol cm -2 s -1 ( = 44) with peritubular capillary control perfusion, 1.88 ± 0.16 nmol cm -2 s -1 ( = 50) during peritubular perfusion, increased to 4.10 ± 0.22 nmol cm -2 s -1 ( = 50) during capillary perfusion with II and decreased to 1.56 ± 0.11 nmol cm -2 s -1 ( = 34) when II plus were added to the peritubular perfusion. Therefore, these studies confirmed that II stimulates proximal bicarbonate reabsorption and showed that alone does not affect this process, but impairs the stimulation caused by II. We have recently studied the effects and interaction of these hormones also in cortical distal nephron acidification (16). Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular ph was measured with a double-barreled microelectrode with one barrel filled with H -sensitive resin and JHO 3 - (nmol cm -2 s -1 ) Early distal 27 the other with 1 M Kl (). Figure 2 shows a significant increase in HO 3 - reabsorption in the presence of luminal II (1 pm) both in ED (from 0.93 ± 0.06, = 20, to 2.64 ± 0.21 nmol cm -2 s -1, = 24) and LD segments (from ± 0.086, =, to 2.16 ± nmol cm -2 s -1, = ). (1 µm) alone did not affect bicarbonate reabsorption in either the ED or LD segment nor did it impair the stimulation caused by II. The addition of the AT 1 receptor antagonist losartan (1 µm) to luminal perfusion blocked luminal II-mediated stimulation in ED and LD segments. We also examined the specific mechanisms by which luminal II stimulates bicarbonate reabsorption. Figure 3 summarizes the results obtained in ED and LD segments during luminal perfusion with II plus hexamethylene-amiloride (HMA, 100 µm, a specific blocker of a /H exchange) or with II plus bafilomycin A1 ( Late distal 33 Figure 2 - Effect of atrial natriuretic peptide (, 1 µm) and/or angiotensin II (, 1 pm) on HO 3 - reabsorption in early (left) and late (right) distal tubules. The experiments were performed in tubules undergoing luminal perfusions with the hormones. Data are reported as means ± SEM., umber of microperfusions; J HO3 -, net HO 3 - reabsorption. P<0.05 compared to control (); P<0.05 compared to (Bonferroni test).

4 474 M. Mello-Aires et al. Figure 3 - Effect of luminal angiotensin II (, 1 pm) plus HMA (100 µm) or bafilomycin A1 (BAF, 200 nm) on HO 3 - reabsorption. The experiments were performed in early (left) and late (right) distal tubules. Data are reported as means ± SEM., umber of microperfusions; J HO3 -, net HO 3 - reabsorption. P<0.05 compared to control (); P<0.05 compared to II (Bonferroni test). JHO 3 - (nmol cm -2 s -1 ) Early distal Late distal HMA 29 BAF 26 HMA BAF Figure 4 - Velocity of ph recovery (dph i /dt) in MDK cells subjected to increasing angiotensin II concentrations (1 pm, 1 nm and 100 nm) alone or in the presence of atrial natriuretic peptide (, 1 µm). The experiments were done in the presence (145 mm) or absence of a. P<0.05 compared to control (); P<0.05 compared to II (Bonferroni test). 0.2 Angiotensin 1 pm 1 nm 100 nm dphi/dt a = 0 mm = 1 µm

5 and II in nephron acidification 475 nm, a highly specific inhibitor of vacuolar H -ATPase). HMA inhibited the stimulation of bicarbonate reabsorption induced by II in ED (J HO3 - decreased to ± nmol cm -2 s -1, = 29) and LD segments (J HO3 - decreased to ± nmol cm -2 s -1, = ). Bafilomycin A1 did not inhibit the stimulation of ED bicarbonate reabsorption induced by II (J HO3 - was 2.50 ± 0.2 nmol cm -2 s -1, = 26); however, in LD segments J HO3 - was significantly decreased to 1.45 ± nmol cm -2 s -1 ( = ). In conclusion, these results indicate that II acts by stimulating a /H exchange in ED (81% of the II-stimulated rate) and LD (54%) segments via activation of AT 1 receptors, as well as vacuolar H -ATPase in LD segments (33%). does not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, does not impair the stimulation caused by II. This result is in accordance with data indicating that in ED segments bicarbonate reabsorption is mediated predominantly by a /H exchange and in LD segments by vacuolar H -ATPase (,18) and with studies that indicate the absence of receptors in distal segments (19). The present study, however, does not allow us to ascribe the results with II to any specific cell type that might contribute to acidification in cortical distal tubules. In order to investigate the mechanism of action of these hormones in more detail, we studied the dependence of cell ph on II and in MDK cells, a permanent cell line derived from dog kidneys, with some properties of distal nephron epithelia. ells were grown to confluence on coverslips, and cytosolic ph was determined with the fluorescent probe BEF in spectrofluorimeter cuvettes. The cells were acid loaded using the H 4 l pre-pulse technique, and the rate of cell ph recovery was followed with control or hormone-containing solutions. Some of the results obtained in these [a 2 ]i (nm) [a 2 ]i (nm) Log II (M) 1 min 1 min II Log peptide (M) studies are illustrated in Figure 4. The velocity of cell ph recovery (dph/dt) was almost abolished in the absence of a, indicating that it is dependent on a /H exchange. This recovery was markedly increased when II was used at concentrations from 1 pm to 1 nm. On the other hand, when concentrations of 100 nm II were used this recovery rate was significantly reduced. We also showed that (1 µm) alone had no effect on this recovery but reversed both the acceleration of H extrusion at low II levels and inhibition of H extrusion at higher II levels. To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [a 2 ] i, monitored with the fluorescent probe Fura-2 in MDK cells in suspension (20). The results are shown in Figure 5. [a 2 ] i increased progressively from control values of 100 nm to approximately 250 nm when II concentration in the cell suspension increased from 1 pm to 1 µm. On the other hand, when 1 µm was added to the cell suspension, [a 2 ] i decreased from control values to 35 nm. The addition of II (1 pm-1 µm) in the presence of increased [a 2 ] i, but without exceeding normal control values. These results may indicate a role of [a 2 ] i in the regulation of the Figure 5 - ell calcium levels in MDK cells subjected to increasing angiotensin II ( II) concentrations (upper panel) alone or in the presence of atrial natriuretic peptide (, 1 µm; lower panel). Data from Ref. 20, with permission.

6 476 M. Mello-Aires et al. References process of H extrusion mediated by a /H exchange and stimulated/impaired by II. The data are compatible with stimulation of a /H exchange by increases of [a 2 ] i in the lower range, and inhibition at high [a 2 ] i levels. At lower levels of II (1 pm-1 nm), the increase in [a 2 ] i may be mediated by AT 1B II receptors which activate phospholipase, causing the stimulation of inositol triphosphate and diacylglycerol, which elevate [a 2 ] i by releasing it from cell stores. alcium activates protein kinase which, via phosphorylation, may stimulate the a / H exchanger (21). Another possible mechanism of action of low-dose II is a signalling pathway involving the activation of an inhibitory G protein and inhibition of adenosylate cyclase, causing a decrease in camp levels and in the catalytic activity of protein kinase A, a reduction which causes the activation of a /H exchange (22). On the other hand, has been shown to block the activity of phospholipase, thus impairing the pathway causing the increase in cell calcium (23). At high levels (100 nm) II interacts with AT 1A receptors, causing the liberation of arachidonic acid, which is part of a pathway that elevates [a 2 ] i by activating voltage-sensitive calcium channels in the plasma membrane (21). At high cytosolic concentrations, calcium may inhibit a /H exchange by activating a /a 2 exchange at the cell membrane level, thereby increasing cell sodium, which in turn decreases the gradient responsible for H extrusion by the exchanger. However, this mechanism is somewhat questionable considering the large discrepancy between cytosolic calcium and extracellular sodium concentrations. On the other hand, it has been shown that the HE1 exchanger has calmodulin-binding sites at the cytoplasmic regulatory domain, which modulate its activity. A highaffinity site, which is tonically inhibitory, binds to low calcium/calmodulin suppressing the inhibition, i.e., stimulating the exchanger at low cell a 2 /calmodulin levels. A low-affinity site, however, binds to calcium and calmodulin only at high concentrations, and under these conditions inhibits the exchanger activity (24). This behavior is compatible with our findings about the II- interaction in proximal nephron and MDK cells. 1. Beasley D & Malvin RL (1985). Atrial extracts increase glomerular filtration rate in vivo. American Journal of Physiology, 248 (Renal, Fluid and Electrolyte Physiology, ): F24-F. 2. ogan MG (1986). Atrial natriuretic factor can increase renal solute excretion primarily by raising glomerular filtration. American Journal of Physiology, 250 (Renal, Fluid and Electrolyte Physiology, 19): F710-F Sosa RE, Volpe M, Marion J, Vaughan ED, Atlas SA, Laragh JH & Maack T (1986). Relationship between renal hemodynamics and natriuretic effects of atrial natriuretic factor. American Journal of Physiology, 250 (Renal, Fluid and Electrolyte Physiology, 19): F520-F Wang T & han YL (1990). holinergic effect on rat proximal convoluted tubule. Pflügers Archiv, 415: Liu FY & ogan MG (1987). Angiotensin II: a potent regulator of acidification in the rat early proximal convoluted tubule. Journal of linical Investigation, 80: Geibel J, Giebisch G & Boron WF (1990). Angiotensin II stimulates both a /H exchange and a /HO 3 - cotransport in the rabbit proximal tubule. Proceedings of the ational Academy of Sciences, USA, 87: oppola S & Frömter E (1994). An electrophysiological study of angiotensin II regulation of a -HO 3 - cotransport and K conductance in renal proximal tubules. I. Effect of picomolar concentrations. Pflügers Archiv, 427: oppola S & Frömter E (1994). An electrophysiological study of angiotensin II regulation of a -HO 3 - cotransport and K conductance in renal proximal tubules. II. Effect of micromolar concentrations. Pflügers Archiv, 427: Kleinert HD, Maack T, Atlas SA, Januszewicz A, Sealey JE & Laragh JH (1984). Atrial natriuretic factor inhibits angiotensin-, norepinephrine-, and potassium-induced vascular contractility. Hypertension, 6 (Suppl I): I.143-I Di icolantonio R, Stevens J, Weaver D & Morgan TO (1986). aptopril antagonizes the hypotensive action of atrial natriuretic peptide in anaesthetized rat. linical and Experimental Pharmacology and Physiology, 13: Atarashi K, Mulrow PJ, Franco-Saenz R, Snajdar R & Rapp J (1984). Inhibition of aldosterone production by an atrial extract. Science, 224: Harris PJ, Thomas D & Morgan TO (1987). Atrial natriuretic peptide inhibits angiotensin-stimulated proximal tubular sodium and water reabsorption. ature, 326:

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